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1.
Complex formation between poly(U) and adenosine in solutions of salts that stabilize (Na2SO4), destabilize (NaClO4), or have little effect on the water structure (NaCl), as well as the poly(U)·poly(A) interaction in NaClO4, was studied by equilibrium dialysis and uv spectroscopy. At 3°C and neutral pH, Ado·2 poly(U) is formed in 1M NaCl and 0.33M Na2SO4. In NaClO4 solutions under the same conditions, an Ado·poly(U) was found over the whole range of salt concentration investigated (10 mM?1M), which has not been previously observed under any conditions. The Ado-poly(U) was also found in a NaCl/NaClO4 mixture, the transition from the triple- to the double-helical complex occurring within a narrow range of concentration of added NaClO4. In the presence of 1M NaCl this transition is observed on adding as little as 10 mM NaClO4, i.e., at a [ClO]/[Cl?] ratio of about 1:100. However, when NaClO4 is added to a 1M solution of the stabilizing salt Na2SO4, no transition occurs even at a [ClO]/[SO] ratio of 1:4. Investigation of melting curves and uv spectra has shown that in an equimolar mixture of the polynucleotides, only a double-helical poly(U)·poly(A) exists in 1M NaClO4 at low temperatures; this also holds for 1M NaCl. This changes to a triple-helical 2 poly(U)·poly(A) and then dissociates as the temperature increases. At low temperatures and the poly(U)/poly(A) concentration ratio of 2:1, a mixture of 2 poly(U)·poly(A) and poly(U)·poly(A) was observed in 1M NaClO4, in contrast to the case of 1M NaCl. Thus, sodium perchlorate, a strong destabilizer of water structure, promotes formation of double-helical complexes both in the polynucleotide–monomer and the polynucleotide–polynucleotide systems. Beginning with a sufficiently high ionic strength (μ ? 0.9), a further increase in the salt molarity results in an increase of the poly(U)·adenosine melting temperature in both stabilizing and neutral salts and a decrease in the destabilizing salt. In Na2SO4 concentrations higher than 1.2M Ado·2 poly(U) precipitates at room temperature. Analysis of the binding isotherms and melting profiles of the complexes between poly(U) and adenosine according to Hill's model shows that the cooperativity of binding, due to adenosine stacking on poly(U), increases in the order NaClO4 < NaCl < Na2SO4. The free energy of adenosine stacking on the template is similar to that of hydrogen bonding between adenosine and poly(U) and ranges from ?1 to ?2 kcal/mol. The values of ΔHt [the effective enthalpy of adenosine binding to poly(U) next to an occupied site, obtained from the relationship between complex melting temperature and free monomer concentration at the midpoint of the transition] are ?14.2, ?18.3, and ?16.8 kcal/mol for 1M solutions of NaClO4, NaCl, and Na2SO4, respectively. The results indicate that the effects of anions of the salts studied are related to water structure alterations rather than to their direct interaction with the complexes between poly(U) and adenosine. 相似文献
2.
Ramsay Richmond Barry Halliwell Jasbir Chauhan Andre Darbre 《Analytical biochemistry》1981,118(2):328-335
A mixture of xanthine or hypoxanthine and xanthine oxidase generates the superoxide radical, O2, and H2O2. In the presence of iron salts, O2 and H2O2 can interact to produce the hydroxyl radical, OH·. Superoxide-dependent formation of OH· can be measured by its ability to hydroxylate salicylate as followed by an improved colorimetric assay described in this paper. A more accurate analysis of OH· can be obtained using its ability to hydroxylate phenol, the hydroxylated products being separated and measured after derivatization using gas-liquid chromatography and electron-capture detection. The derivatization and separation techniques are described. 相似文献
3.
Daucus carota cells contain a dihydrofolate reductase: thymidylate synthase bifunctional polypeptide
Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) activities from cell suspension cultures of Daucus carota were shown to copurify on (NH4)2SO4 fractionation, DEAE Sephadex and methotrexate-Sepharose affinity chromatography and to share approximately the same Mr(183 kDa and 185 kDa respectively) as judged by gel filtration on Sephacryl S-200.The copurified protein migrated as a single band on polyacrylamide gel electrophoresis under denaturing conditions.Both activities could be eluted from the same position of the native gel.Moreover, methotrexate-resistant cell lines which overproduce DHFR revealed to have a parallel higher level of TS. It is therefore proposed and discussed that in carrot, similarly to protozoa, TS and DHFR are present on a single bifunctional polypeptide of 58 kDa. 相似文献
4.
Summary The benzoyl-CoA ligase from an anaerobic syntrophic culture was purified to homogeneity. It had a molecular mass of around 420 kDa and consisted of seven or eight subunits of 58 kDa. The temperature optimum was 37–40° C, the optimum pH around 8.0 and optimal activity required 50–100 mM TRIS-HCI buffer, pH 8.0 and 3–7 mM MgCl2; MgCl2 in excess of 10 mM was inhibitory. The activation energy for benzoate was 11.3 kcal/mol. Although growth occured only with benzoate as a carbon source, the benzoyl-coenzyme A (CoA) ligase formed benzoyl-CoA esters with benzoate, 2-, 3- and 4-fluorobenzoate, picolinate, nicotinate and isonicotinate. Acetate was activated to acetyl-CoA by an acetyl-CoA synthetase. The K
m values for benzoate, 2-, 3- and 4-fluorobenzoate were 0.04, 0.28, 1.48 and 0.32 mM, the V
max values 1.05, 1.0, 0.7 and 0.98 units (U)/mg, respectively. For reduced CoA (CoA-SH) a K
m of 0.17 mM and a V
max of 1.05 U/mg and for ATP a K
m of 0.16 mM and a V
max of 1.08 U/mg was determined. Benzoate activation was inhibited by more than 6 mM ATP, presumably by pyrophosphate generation from ATP. The inhibition constant (K
i) for pyrophosphate was 5.7 mM. No homology of the N-terminal amino acid sequence with that of a 2-aminobenzoyl-CoA ligase of a denitrifying Pseudomonas sp. was found.
Correspondence to: J. Winter 相似文献
5.
G. J. Grobben W. H. M. van Casteren H. A. Schols A. Oosterveld G. Sala M. R. Smith J. Sikkema J. A. M. de Bont 《Applied microbiology and biotechnology》1997,48(4):516-521
The exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in defined medium were investigated. At equal cell densities, the strain produced 95 mg l−1 exopolysaccharides with glucose and 30 mg l−1 with fructose as the carbohydrate source. High-performance size-exclusion chromatography of the exopolysaccharides produced
on glucose showed the presence of two fractions with relative molecular masses (M
r) of 1.7 × 106 and 4 × 104 in almost equal amounts. The exopolysaccharides produced on fructose contained mainly a fraction of low M
r of 4 × 104. The high-M
r fraction of the purified exopolysaccharides produced on glucose appeared to have a sugar composition of galactose, glucose
and rhamnose in the molar ratio of 5:1:1, whereas the low-M
r weight fraction contained galactose, glucose and rhamnose in the molar ratio of approximately 11:1:0.4. The purified exopolysaccharide
fractions produced on fructose showed comparable ratios. The high-molecular-mass fractions contained terminally linked galactose,
1,2,3-linked galactose, 1,3,4-linked galactose, 1,3-linked glucose and terminally linked rhamnose. The low-molecular-mass
fractions contained mainly 1,3-linked galactose and 1,6-linked galactose and lower amounts of other sugar linkages. The production
of the high-M
r fractions appeared to be dependent on the carbohydrate source, whereas the low-M
r fractions were produced more continuously.
Received: 30 April 1997 / Received revision: 11 June 1997 / Accepted: 14 June 1997 相似文献
6.
Indalecio Quesada‐Soriano Lorien J. Parker Alessandra Primavera Juan M. Casas‐Solvas Antonio Vargas‐Berenguel Carmen Barón Craig J. Morton Anna Paola Mazzetti Mario Lo Bello Michael W. Parker Luis García‐Fuentes 《Protein science : a publication of the Protein Society》2009,18(12):2454-2470
The effect of the Y108V mutation of human glutathione S‐transferase P1‐1 (hGST P1‐1) on the binding of the diuretic drug ethacrynic acid (EA) and its glutathione conjugate (EASG) was investigated by calorimetric, spectrofluorimetric, and crystallographic studies. The mutation Tyr 108 → Val resulted in a 3D‐structure very similar to the wild type (wt) enzyme, where both the hydrophobic ligand binding site (H‐site) and glutathione binding site (G‐site) are unchanged except for the mutation itself. However, due to a slight increase in the hydrophobicity of the H‐site, as a consequence of the mutation, an increase in the entropy was observed. The Y108V mutation does not affect the affinity of EASG for the enzyme, which has a higher affinity (Kd ~ 0.5 μM) when compared with those of the parent compounds, K ~ 13 μM, K ~ 25 μM. The EA moiety of the conjugate binds in the H‐site of Y108V mutant in a fashion completely different to those observed in the crystal structures of the EA or EASG wt complex structures. We further demonstrate that the ΔCp values of binding can also be correlated with the potential stacking interactions between ligand and residues located in the binding sites as predicted from crystal structures. Moreover, the mutation does not significantly affect the global stability of the enzyme. Our results demonstrate that calorimetric measurements maybe useful in determining the preference of binding (the binding mode) for a drug to a specific site of the enzyme, even in the absence of structural information. 相似文献
7.
P. Khanna S. Sivakami Sundari N. Jothi Kumar 《World journal of microbiology & biotechnology》1995,11(2):242-243
AnAspergillus sp., isolated from a rubbish dump, produced 10.6 IU ml-1 xylanase activity. Two xylanases were recognized and each was purified to homogeneity by two-stage chromatography on DEAE-and CM-Sepharose. Xylanase I had a pI of 7.2 and anM
r of 26 kDa whereas xylanase II had a pI of 4.7 and anM
r of 21 kDa. At 50°C, xylanase I was stable for 2.5 h but xylanase II was only stable for 1 h.P. Khanna is with the National Environmental Engineering Research Institute, Nehru Marg, Nagpur 440 020, India. S. Sivakami Sundari and N. Jothi Kumar are with the National Environmental Engineering Research Institute, Madras Zonal Laboratory, CSIR Madras Complex, Taramani 600 113, India. 相似文献
8.
Molecular conformation of some polysaccharides in aqueous solution in evidenced by changes in the optical rotation and in the elution pattern of gel filtration. The changes in the specific rotation against that in water are expressed as a molar conformational value [K]: ?495° for colominic acid in 1.0 N NaOH solution, and ?180° for hyaluronate (HA), +85° for corneal keratin sulfate, and +234° for amylose in 8 M urea solution. The gel filtration of amylose and HA dissolved in 8 M urea solution shows elution patterns distinctly different from those dissolved in water. The phenomena are attributable to a molecular conformational transition of polysaccharide molecules in aqueous solution. 相似文献
9.
《Bioscience, biotechnology, and biochemistry》2013,77(11):2258-2262
A truncated apolipoprotein (apo) A-I with a molecular weight (M r) of 26 kDa was first isolated from the plasma high density lipoproteins of an atypical Japanese eel (Anguilla japonica). Interestingly, this eel contained a very small amount of intact apoA-I (M r28 kDa) in the plasma, although serine protease inhibitors were present throughout the plasma preparation. The N-terminal sequence of 20 amino acids in truncated apoA-I was completely identical with that of intact apoA-I. Another apolipoprotein with M r28 kDa, whose N-terminal amino acid sequence differed from apoA-I, was also found in high density lipoprotein and low density lipoprotein. The apolipoprotein profiles of Japanese eel plasma appear to be complicated. 相似文献
10.
Katarína Bíliková Ekaterina Mirgorodskaya Gabriela Bukovská Johan Gobom Hans Lehrach Jozef Šimúth 《Proteomics》2009,9(8):2131-2138
This study illustrates multifunctionality of proteins of honeybee royal jelly (RJ) and how their neofunctionalization result from various PTMs of maternal proteins. Major proteins of RJ, designated as apalbumins belong to a protein family consisting of nine members with Mr of 49–87 kDa and they are accompanied by high number of minority homologs derived from maternal apalbumins. In spite of many data on diversity of apalbumins, the molecular study of their individual minority homologous is still missing. This work is a contribution to functional proteomics of second most abundant protein of RJ apalbumin2 (Mr 52.7 kDa). We have purified a minority protein from RJ; named as apalbumin2a, differ from apalbumin2 in Mr (48.6 kDa), in N‐terminal amino acids sequences – ENSPRN and in N‐linked glycans. Characterization of apalbumin2a by LC‐MALDI TOF/TOF MS revealed that it is a minority homolog of the major basic royal jelly protein, apalbumin2, carrying two fully occupied N‐glycosylation sites, one with high‐mannose structure, HexNAc2Hex9, and another carrying complex type antennary structures, HexNAc4Hex3 and HexNAc5Hex4. We have found that apalbumin2a inhibit growth of Paenibacillus larvae. The obtained data call attention to functional plasticity of RJ proteins with potential impact on functional proteomics in medicine. 相似文献
11.
A new bacterial isolate, 00-50-5, from sunflower head extracts was identified as Bacillus thuringiensis (Bt) according to its morphology. Bt isolate 00-50-5 was highly active against the banded sunflower moth (BSM), Cochylis hospes Walsingham. A sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 4–15% gradient gel of whole strain protein
of 00-50-5 revealed six proteins with molecular masses (Mr) of 133, 80, 60, 27, 15, and 14 kDa. SDS-PAGE of pH 4.2-precipitated proteins (PP) or activated proteins formed by adding
the BSM larval gut protease at 1:50 (wt/wt, protease/PP) showed five bands, including two major proteins of Mr 60 kDa and 27 kDa, and three small peptides of Mr 15, 13, and 7 kDa. The BSM larval gut protease was able to completely digest the proteins when present at a high ratio (10:1,
wt/wt, protease/PP). The 60- and 27-kDa proteins could be digested by subtilisin Carlsberg at ratios of 1:50 or 1:1 (wt/wt,
protease/PP), but neither BSM larval gut protease nor trypsin was effective at the same ratios. Three small peptides of Mr 15, 13, and 7 kDa were digested by the gut protease at a ratio of 1:1 (wt/wt). The N-terminal sequence of 1–31 amino acid
residues for the 27-kDa protein showed 96.7% homology to a 31-amino acid fragment from camelysin, a protease from B. cereus, indicating that the 27-kDa protein may be a camelysin and a novel active protein against BSM.
Received: 9 July 2001 / Accepted: 8 August 2001 相似文献
12.
Aims: To isolate a fucoidan‐utilizing strain from seawater for sea cucumber fucoidan degradation. Methods and Results: The utilization of sea cucumber fucoidan was monitored by H2SO4–phenol assay for neutral sugar. The bacterium CZ1127 was isolated from seawater and shown to have a relatively large maximum fucoidan‐utilizing rate of 81·5%. CZ1127 was confirmed to belong to the family Flavobacteriaceae by 16S rDNA and physiological analyses. This strain has an ability to utilize fucoidans extracted from various sea cucumbers to different degrees. Both extracellular and intracellular enzymes of CZ1127 could degrade sea cucumber fucoidan, as confirmed by high‐performance size exclusion chromatography. The Mr of sea cucumber fucoidan could be reduced from 792·6 kDa to at least 3·7 kDa by the crude intracellular enzyme of this strain. Conclusions: The marine bacterial strain CZ1127, which belongs to the family Flavobacteriaceae, was found to utilize various sea cucumber fucoidans and furthermore showed promise in sea cucumber fucoidan enzymatic degradation and oligosaccharide preparation. Significance and Impact of the Study: The finding of a novel source can be applied in sea cucumber fucoidan enzymatic degradation. Furthermore, it is the first definite report of a bacterial strain that can utilize the fucoidans from various sea cucumbers. 相似文献
13.
Purification and characterization of microsomal glutathione S-transferase produced by Aspergillus ochraceus TS are reported. The isozymes are located in microsomes and were active against 1-chloro-2,4-dinitrobenzene, ethacrynic acid, 1,2-dichloro-4-nitrobenzene, trans-4- phenyl-3-buten-2-one,p-nitrobenzyl chloride and bromosulphophthalein. They were inhibited by N-ethylmaleimide and bromosulphophthalein. The GST isozymes produced by Aspergillus ochraceus TS are indistinguishable in respect of their molecular mass both in native and denatured state. The subunit of the purified protein had an apparent Mr of 11 kDa while molecular mass of the native protein is around 56 kDa. The substrate specificity and pl values of the isozymes were different. The GSTs produced by Aspergillus ochraceus TS fairly share functional properties with mammalian cytosolic isozymes. 相似文献
14.
Equilibrium unfolding (folding) studies reveal that the autoregulatory RNA pseudoknots derived from the bacteriophage T2 and T4 gene 32 mRNAs exhibit significant stabilization by increasing concentrations of divalent metal ions in solution. In this report, the apparent affinities of exchange inert trivalent Co(NH3) have been determined, relative to divalent Mg2+, for the folded, partially folded (Kf), and fully unfolded (Ku) conformations of these molecules. A general nonspecific, delocalized ion binding model was developed and applied to the analysis of the metal ion concentration dependence of individual two‐state unfolding transitions. Trivalent Co(NH3) was found to associate with the fully folded and partially unfolded pseudoknotted forms of these RNAs with a Kf of 5–8 × 104 M−1 in a background of 0.10 M K+, or 3‐ to 5‐fold larger than the Kf obtained for two model RNA hairpins and hairpin unfolding intermediates, and ≈ 40–50‐fold larger than Kf for Mg2+. The magnitude of Kf was found to be strongly dependent on the monovalent salt concentration in a manner qualitatively consistent with polyelectrolyte theory, with Kf reaching 1.2 × 105 M−1 in 50 mM K+. Two RNA hairpins were found to have affinities for Co(NH3) and Ru(NH3) of 1–2 ×104 M−1, or ≈ 15‐fold larger than the Kf of ∼ 1000 M−1 observed for Mg2+. Additionally, the Ku of 4,800 M−1 for the trivalent ligands is ≈ 8‐fold larger than the Ku of 600 M−1 observed for Mg2+. These findings suggest that the T2 and T4 gene 32 mRNA pseudoknots possess a site(s) for Mg2+ and Co(NH3) binding of significantly higher affinity than a “duplexlike” delocalized ion binding site that is strongly linked to the thermodynamic stability of these molecules. Imino proton perturbation nmr spectroscopy suggests that this site(s) lies near the base of the pseudoknot stem S2, near a patch of high negative electrostatic potential associated with the region where the single loop L1 adenosine crosses the major groove of stem S2. © 1999 John Wiley & Sons, Inc. Biopoly 50: 443–458, 1999 相似文献
15.
Kamila Rzeznicka Sebastian Schätzle Dominique Böttcher Joachim Klein Uwe T. Bornscheuer 《Applied microbiology and biotechnology》2010,85(5):1417-1425
The nitrile hydratase (NHase, EC 4.2.1.84) genes (α and β subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (α subunit, Mr 23 kDa) and 218 amino acids (β subunit, Mr 24 kDa) and the NHase activator of 413 amino acids (Mr 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly
in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator
gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration
and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic
ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity
was observed for aromatic compounds only with E values ranging 5–17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30°C and showed the highest
stability at 4°C in thermal inactivation studies between 4°C and 50°C. 相似文献
16.
Changes in Δν½, the nmr linewidth of 23Na, have been determined during titrations of helical DNA with polyamines (divalent putrescine and trivalent spermidine) and with inorganic cations (Mg2+ and Co(NH3)). In each case additions of a multivalent cation (Mz+) to a solution containing NaDNA and NaCl cause decreases in Δν½, which is a population-weighted average of contributions from nuclei in bound and free environments. Thus, the binding of Mz+ to DNA displaces sodium ions from regions where the quadrupolar relaxation of 23Na is relatively efficient. At a given extent of titration, the binding of a polyamine produces a smaller decrease in Δν½ than does the binding of an inorganic ion of the same valence. The concentration dependence of Δν½ during the course of a titration can be interpreted most simply as a two-state ion-exchange reaction by assuming that the binding of Mz does not alter RB, the average relaxation rate of sodium nuclei that remain bound. On the basis of this assumption, the initial linear portions of titration curves can be analyzed to determine upper bounds for r°, the number of sodium ions bound per DNA phosphate in the absence of any competing counterion. Analyzing the titration curves for the four multivalent competitors leads to a range of upper-bound estimates for r°: 0.5–0.8. The differences in these estimates could indicate that polyamines displace fewer sodium ions from DNA than do their smaller inorganic counterparts. Alternatively, the range in upper-bound estimates for r° could also reflect specific differences in the effects of the various multivalent cations on RB, if this relaxation rate does change during titration. 相似文献
17.
A protein, identifiable as calmodulin (CaM), has been isolated from the seedling tissue of Pharbitis nil. The method has been developed to isolate a high quality protein from plant tissue containing the high content of polyphenols. This protein was relatively heat-stable and bound to hydrophobic resin in calcium-dependent manner. It was recognized by the antibody against pea and carrot, but did not bind to antibody against Dictyostelium discoideum. This protein had Mr of 15 kDa and 18.5 kDa in the presence and absence of Ca2+, respectively, and was able to stimulate calmodulin-deficient cAMP phosphodiesterase. Based on its migration on SDS-PAGE gels, Mr and binding to anti-CaM antibodies it was deduced that calmodulin from P. nil is essentially identical to calmodulin isolated from other plants. 相似文献
18.
Mass‐specific oxygen consumption rate, i.e. standard metabolic rate (Rs) and critical oxygen tension (Pcrit) of red drum Sciaenops ocellatus were measured and scaled over a 2500‐fold range in mass (MF; 0·26–686 g). Rs conformed to well established models (Rs = 3·73·91 MF?0·21; r2 = 0·86) while Pcrit increased over the size range (Pcrit = 3·15 log10MF + 16·19; r2 = 0·44). This relationship may be ecologically advantageous as it would allow smaller S. ocellatus to better utilize hypoxic zones as habitat and refuge from predators. 相似文献
19.
Thomas Kunze 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1997,116(4):397-406
Six cytosolic GSTs from porcine liver were purified by a combination of glutathione affinity chromatography and ion-exchange HPLC. The isoenzymes were characterized by SDS-PAGE, gel filtration, isoelectric focusing, immunoblotting analysis and determination of substrate specificities and inhibition characteristics. The purified GSTs belong to the alpha and mu classes, respectively. No class pi isoenzyme was isolated or detected. The class alpha GST pA1-1* exists as a homodimer (Mr = 25.3 kDa), whereas GST pA2-3* consists of two subunits with different Mr values (27.0 and 25.3 kDa). The estimated pI values were 9.5 and 8.8, respectively. Furthermore, four class mu porcine GSTs, pM1-1*, pM1-2*, pM3-?* and pM4-?*, were isolated. The isoenzyme pM1-1* possesses a relative molecular mass of 27.2 kDa and a pI value of 6.2. Additional pM1 isoenzymes hybridize with the subunit pM2* (Mr = 25.2) to furnish a heterodimer, which shows a pI value of 5.8. The other class mu isoenzymes are heterodimers with pI values of 5.45 and 5.05. Substrate specificities and inhibition characteristics correlate very well with those of the corresponding human isoenzymes. The results are discussed with regard to the usefulness of porcine GSTs as an in vitro testing model. 相似文献
20.
The region ofBacteroides fragilis DNA on the recombinant plasmid pMT100 responsible for conferring metronidazole resistance inEscherichia coli strains was characterized. An open reading frame (ORF1) of 195 bp encoded a protein of 64 amino acids with a predicted Mr, of 7.3 kDa. Deletion analysis indicated that ORF1 conferred the metronidazole resistance phenotype and encoded a protein with an apparent Mr of approximately 8–10 kDa. 相似文献